Indian Journal of Experimental Biology Vol. 45, July 2007, pp. 579-593 Review Article Bioactive molecules from amphibian skin: Their biological activities with reference to therapeutic potentials for possible drug development Antony Gomes1, Biplab Giri1, Archita Saha1, R Mishra1, Subir C Dasgupta1, A Debnath2 & Aparna Gomes2 1Laboratory of Toxinology and Experimental Pharmacodynamics Department of Physiology, University of Calcutta 92 A. P. C. Road, Kolkata 700 009, India 2Division of New Drug Development, Indian Institute of Chemical Biology 4 S. C. Mullick Road, Kolkata 700 032, India The amphibian skin contains various bioactive molecules (peptides, proteins, steroids, alkaloids, opiods) that possess potent therapeutic activities like antibacterial, antifungal, antiprotozoal, antidiabetic, antineoplastic, analgesic and sleep inducing properties. Research on amphibian skin derived biomolecules can provide potential clue towards newer drug development to combat various pathophysiological conditions. An overview on the bioactive molecules of various amphibian skins has been discussed. Keywords: Amphibians, Frog skin, Medicinal application, Skin bioactive molecules, Therapeutic potential, Toad skin The amphibians are defenseless creatures that are in traditional medicines. Torched newts are consumed preferably by a great variety of predators. sometimes sold in Asia as aphrodisiacs and the skin of In order to protect themselves from the potential certain species are said to cure illnesses. Superstitions predators, the amphibians have evolved different and folklore as these may be, they were actually the morphological, physiological and behavioral features. stepping-stones to modern biological sciences. One such defense mechanism is the slimy glandular In fact, toad and frog skin extracts have been used secretion by the skin. Frogs and toads have two types in Chinese medicine for treating various ailments. of skin glands–the mucous and the granular. The Chan Su is a traditional Chinese medicine prepared mucous glands occur throughout the skin and their from dried white secretion of the skin glands of secretion provides a moist coating that is necessary Chinese toad and has been used as an oriental drug for for cutaneous respiration. The granular glands, also treating heart diseases, toothache, sinusitis, called the serous or poison glands, may be distributed haemorrhage of gums and other systemic illnesses. across the body but are often concentrated around the Chinese doctors prescribed drinking of wine (in which head or neck, and are usually activated by stress or toad skin has been soaked) for the treatment of injury, and their secretions vary from species to leukemia. A well known, but somewhat toxic patent species from being slightly noxious to extraordinarily medicine, Liu Shen Wan (containing toad secretion toxic1. and realgar), is sometimes recommended by Chinese In many ancient cultures, amphibians are believed doctors for leukemia. Scientific research has to possess medicinal properties. Frog potions are used confirmed the presence of bufalin in Chan Su, which as aphrodisiacs, impotence and infertility preventions, is responsible for digoxin-like action2. Scientists have contraceptives and in many other illnesses. Also, also found that Chan Su possesses antineoplastic newts are often burned to ashes and then used in properties3 and components of Chan Su are capable of medicinal formulas and concoctions. Even today, the potentiating immune responses in experimental skins, bodies and body parts of salamanders are used animals4. Scientists throughout the world are now exploring the therapeutic potential of various toad and _______________ frog skin extracts and secretions. India, being a Phone: +91-33-2350-8386 (Extn 229); Fax: +91-33-2351-9755; +91-33-2241-3288 tropical country with diverse geographical variations, E-mail: [email protected] is home to wide species of toads, frogs and newts. 580 INDIAN J EXP BIOL, JULY 2007 There are about 200 species of frogs and toads in Na-K-ATPase activity12-17. The secretion from Bufo India. However, not much work has been carried out viridis Laur. skin glands produces a marked on toads and frog skin secretions in India as compared cardiotonic and vasotonic effect, increasing the to the rest of the world. Though, the jacket (coat) of intraventricular and aortal pressure, the rate of some of the most toxic frogs and toads might just be pressure growth in the ventricles and the contraction the treasure house, storing some of the best index of the myocardium on experimental dogs under pharmaceutical promises. The present communication anaesthesia18. is an effort to enlighten the importance of amphibian Antidiabetic activity skin – a future treasure house for development of Many insulinotropic peptides have been isolated novel drugs against several pathophysiological from different toad and frog skins. Insulin-releasing conditions. peptides (peaks 21, 22, 23, 24, 25) purified by reverse Cardiotonic and anti-arrhythmic activity phase HPLC from skin secretions of the toad Chan Su is prepared from skin secretion of Chinese Bombina variegata have molecular masses of 1.64, toad (Bufo bufo gargarizans), for treating arrhythmia 1.66, 1.68, 1.65 and 2.30 kDa, respectively19. They and other heart diseases. It had been introduced in significantly increase the insulin release by glucose Europe in 17th century and was later replaced by responsive BRIN-BD11 cells as compared to glucose digitalis some 200 years ago5. Kyushin (Japanese (5.6mM) alone. Peaks 21, 22 and 23 have sequence medicine similar to Chan Su) shows a beneficial homology to bombesin (Pyr-QRLGNQWAVGHLM) effect on congestive heart failure models in rabbits originally isolated from Bombina bombina, while due to its cardiotonic property6,7. The vasodilating peaks 24 and 25 are new. The mechanism underlying effect and positive inotropic action of Kyushin is their insulinotropic actions suggests possible probably due to beta-adrenergic action8. Kyushin involvement of a cAMP dependent G-protein significantly inhibits the aconitine-induced and insensitive pathway19. Fractions purified from skin thyroxine-induced arrhythmia in guinea pigs. The secretion of Rana palustris by reverse-phase HPLC decrease in heart rate induced by electrical stimulation also increased the insulin-releasing activity of BRIN- to the parasympathetic nerve (vagus nerve) can be BD11 cells. One such peptide (2.87 kDa) has a 27 restored by Kyushin, though Kyushin itself does not amino acid sequence, ALSILRGLEKLAKMGIAL- affect the conduction system in Langendorff TNCKATKKC (having 48% homology with preparation of rabbit hearts9. Kyushin dose- antimicrobial peptide Brevinin-120. Insulinotropic dependently increases the left ventricular pressure and peptides are also present in the skin secretions of mean aortic pressure and decreases the left ventricular Phyllomedusa trinitatis frog. One of them−a 28- end-diastolic pressure in a dose-dependent manner, amino-acid peptide had 100% homology with the C- but the myocardial oxygen consumption and heart rate terminal of the 75-amino-acid dermaseptin BIV are not significantly affected in anaesthetized dogs10. precursor in the skin of the Phyllomedusinae Bufalin and cinobufagin are the main components of subfamily21. Insulinotropic peptides were also Chan Su, which are responsible for its digitalis-like secreted by the skin of Agalychnis litodryas22 and action. Although Kyushin itself does not affect the Agalychnis calcarifer frogs23. An insulin-releasing conduction system, bufalin (0.3mg/ml) and peptide isolated from Rana pipiens had 100% cinobufagin (1 mg/ml) inhibit conduction in sequence homology with an antimicrobial peptide Langendorff preparation of isolated rabbit heart9. pipinin-124. Skin secretions of Rana saharica Bufalin, cinobufagin and some other bufadienolides possessed nontoxic insulinotropic peptides25. Two like bufotalin, cinobufotalin, gamabufotalin and were novel 1.89 and 2.93 kDa peptides. Other four resibufogenin⎯all show the cardiotonic effect in a (2.67, 3.51, 4.92 and 4.8 kDa) were identical to concentration-dependent manner in guinea pig brevinin-1E, brevinin-2EC, esculentin-1 and isolated heart preparations and cinobufagin possesses esculentin-1B, which belong to the group of the most cardiotonic action, similar to digitoxin, in antimicrobial peptides isolated from skin secretions of experimentally induced heart failure due to acute various Rana frog species. Contrast to the peptides local ischemia11. The cardiotonic steroids derived from Bombina, the mechanism underlying the from toad venom (bufalin, bufotalin, resibufagenin, insulinotropic actions of esculentins-1 and -1B marinobufagenin, marinoic acid, marinosin) all inhibit possibly involves both cyclic AMP-protein kinase A GOMES et al.: THERAPEUTIC POTENTIAL OF AMPHIBIAN SKIN BIOACTIVE MOLECULES 581 and C dependent G-protein sensitive pathways 26. from the skin secretions of the dusky gopher frog However, all the studies reported were done in vitro. (Rana sevosa), shares 77.8% homology with pLR, can It would be interesting to see if such compounds release histamine from rat peritoneal mast cells and could increase the insulin release in vivo. These inhibits the early development of granulocyte insulinotropic peptides may be exploited as macrophage colonies from bone marrow stem cells antidiabetic agents in the long run. but does not induce apoptosis of the end stage granulocytes31. Immunomodulatory activity The water-soluble, non-dialyzable